Ma et al.

Biotechnol Biofuels (2017) 10:137

DOI 10.1186/s13068-017-0821-1 Biotechnology for Biofuels

RESEARCH Open Access

Novel insight into the relationship

between organic substrate composition
and volatile fatty acids distribution
in acidogenic co‑fermentation
Huijun Ma1, He Liu1,2*, Lihui Zhang1, Meng Yang1, Bo Fu1,2 and Hongbo Liu1,2

Abstract 
Background:  Co-fermentation is an attractive technology for improving volatile fatty acids (VFAs) production by
treatment of solid organic wastes. However, it remains unclear how the composition of different organic matters in
solid waste influences the VFAs distribution, microbial community structure, and metabolic pathway during acido-
genic co-fermentation. In this study, different organic wastes were added into waste activated sludge (WAS) as co-
fermentation substrates to explore the impact of organic matter composition on VFAs pattern and the microbiological
mechanism .
Results:  Acetate was the most dominant VFA produced in all fermentation groups, making up 41.3–57.6% of the
total VFAs produced during acidogenic co-fermentation under alkaline condition. With the increased addition of
potato peel waste, the concentrations of propionate and valerate decreased dramatically, while ethanol and butyrate
concentrations increased. The addition of food waste caused gradual decreases of valerate and propionate, but etha-
nol increased and butyrate was relatively stable. Some inconsistency was observed between hydrolysis efficiency and
acidification efficiency. Our results revealed that starch was mainly responsible for butyrate and ethanol formation,
while lipids and protein favored the synthesis of valerate and propionate. Microbial community analysis by high-
throughput sequencing showed that Firmicutes had the highest relative abundance at phylum level in all fermenta-
tion groups. With 75% potato peel waste or 75% food waste addition to WAS, Bacilli (72.2%) and Clostridia (56.2%)
were the dominant respective classes. In fermentation using only potato peel waste, the Bacilli content was 64.1%,
while the Clostridia content was 53.6% in the food-only waste fermentation.
Conclusions:  Acetate was always the dominant product in acidogenic co-fermentation, regardless of the substrate
composition. The addition of carbon-rich substrates significantly enhanced butyrate and ethanol accumulation, while
protein-rich substrate substantially benefited propionate and valerate generation. Potato peel waste substantially
favored the enrichment of Bacilli, while food waste dramatically increased Clostridia content in the sludge.
Keywords:  Waste activated sludge, Substrate composition, VFA distribution, Microbial community, Acidogenic
co-fermentation, Alkaline pH

Background are now aimed toward resource recovery. Common tar-

With increasing energy demands and a shortfall in
renewable resources, many studies of energy utilization
*Correspondence: liuhe@jiangnan.edu.cn
1
School of Environmental and Civil Engineering, Jiangnan University,
Wuxi 214122, China
Full list of author information is available at the end of the article
gets include biogas and volatile fatty acids (VFAs) from
agricultural residues, food waste, and sewage sludge
containing high levels of organic matter [1, 2]. The large
amounts of proteins, polysaccharides, and other types of
organic matter in waste activated sludge (WAS) can be
efficiently hydrolyzed to water-soluble organic substances

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ma et al. Biotechnol Biofuels (2017) 10:137 Page 2 of 15

by fermentative microorganisms, thus achieving resource experimental conditions were studied by high-through-
recovery [3, 4]. VFAs, the main intermediates during put sequencing analysis.
anaerobic fermentation, have been recognized as a valu-
able carbon resource in wastewater, and can be used as Methods
platform molecules to produce biopolymers and medium Characteristics of WAS, potato peel waste, and food waste
chain fatty acids with high added value [5–7]. In addi- WAS used in fermentation was dewatered sludge
tion to the improvement of VFA yields during anaerobic obtained from a municipal wastewater treatment plant
fermentation of solid wastes [3, 8], it is also important to in Wuxi, China (Shuofang wastewater treatment plant,
control the composition of VFAs and to achieve selective Wuxi, Jiangsu). Potato peel waste was obtained from
VFA production to benefit downstream processing and a vegetable market in Wuxi, and food waste was col-
applications. lected from the canteen of Jiangnan University. The food
An increasing number of studies are now using co- waste mainly consisted of fat, rice, meat, and vegetables.
fermentation to improve the VFAs product yield from Potato peel waste and food waste were fully crushed by
anaerobic sludge fermentation. However, there is little a food crusher after bones and other hard objects were
information on how the complex organic matter content removed. WAS, crushed potato peel waste, and crushed
influences the VFAs distribution and the microbial mech- food waste were stored at 4  °C for subsequent fermen-
anism that proceeds during co-fermentation. For exam- tation experiments. Composition and properties of the
ple, VFAs yields were 1.75-, 10.7-, and 2.6-fold higher wastes are shown in Table 1. The lipid content in potato
than WAS fermentation alone, when food waste, peren- peel waste was below the detection limit, and its con-
nial ryegrass, or henna plant was used as co-fermentation centration was neglected in the following calculations of
substrate, respectively, with a carbon-to-nitrogen ratio organic consumption.
(C/N) of about 22/1 [9–11]. Interestingly, reported pat- Seeding sludge was obtained from the Shuofang waste-
terns of VFAs in previous co-fermentation studies were water treatment plant, and an upflow anaerobic sludge
different, even when the substrate types and addition blanket (UASB) reactor (effective volume  ~5.0  L) was
ratios were similar. Reported VFAs yields were twofold used for acidogenic bacteria acclimation. The WAS
[12] and 10.6-fold [13] higher than for sludge fermenta- was heated at 105  °C for 2  h to kill non-spore-forming
tion alone when the mixing ratio of food waste to sludge methanogens. Before being used as sludge inoculum,
was 50% during co-fermentation. Some studies have the heat-treated sludge was added to the UASB reac-
attributed the increased VFAs yields to the involvement tor for re-activation. Glucose was continuously pumped
of organic matter and the microbial community [14, 15]. into the UASB to enrich the acidogenic bacteria during
For example, when adding different types of substrates, the operation, and the temperature was maintained at
some major phyla, such as Firmicutes, Chloroflexi, and 37  ± 2 °C. Seeding sludge was obtained when the efflu-
Proteobacteria, were significantly changed [16]. ent pH fell below 4.0. The cultivation period was more
These previous studies hinted that the composition than 3  weeks [17]. Characteristics of the seeding sludge
of the organic matter in solid wastes significantly influ-
enced the composition of the fermented VFAs. However,
Table 1 Characteristics of  WAS, potato peel waste,
research has been lacking on the detailed relationship
and food waste
between the organic matter and the distribution of VFAs,
and on how the organic composition influences the Parameters WAS Potato peel Food waste
waste
microbial community or metabolic pathway. This knowl-
edge gap has hindered the industrial application of selec- Solid content (%) 14.54 ± 0.10 20.59 ± 1.42 35.12 ± 1.03
tive VFA production process. VS/TS (%) 45.85 ± 0.29 94.62 ± 0.61 93.68 ± 0.47
This study investigated the effects of different organic pH 6.60 ± 0.08 6.01 ± 0.12 5.08 ± 0.16
matters on the VFAs metabolic pathway and the micro- TCOD (mg/g TS) 849.29 ± 19.45 1292.45 ± 28.94 1497.32 ± 39.23
bial community shift during the co-fermentation of SCOD (mg/g TS) 262.78 ± 6.41 543.20 ± 2.02 618.28 ± 7.13
sludge and carbon-rich organic wastes. Potato peel TN (mg/g TS) 25.90 ± 1.24 4.53 ± 0.07 10.53 ± 1.06
waste and food waste, the two common carbon-rich sub- Protein (mg/g VS) 323.96 ± 5.86 69.81 ± 5.60 123.03 ± 2.56
strates used in China, were added into WAS in differ- Carbohydrate 305.34 ± 12.32 792.23 ± 10.34 284.65 ± 5.54
ent proportions for anaerobic fermentation. The release (mg/g VS)
and consumption of different organic components were Starch (mg/g VS) 183.74 ± 3.12 673.44 ± 4.36 214.50 ± 1.34
studied along with the VFAs product yields and compo- Lipid (mg/g VS) 93.73 ± 0.45 * 564.23 ± 1.54
sition changes. Shifts in microbial community structure VFAs (mg/g VS) 18.27 ± 1.20 20.82 ± 1.35 27.94 ± 1.84
and functional community evolution under the different * Not detected
 Ma et al. Biotechnol Biofuels (2017) 10:137

Table 2  Sample set-up and concentrations of organic materials before fermentation

Fermen‑ OS OP OF SP1 SP2 SP3 SF1 SF2 SF3
tation
groups

Total VS 30.25 ± 0.54 32.03 ± 1.02 30.33 ± 0.72 28.01 ± 0.20 29.63 ± 0.57 30.43 ± 0.89 31.46 ± 1.06 29.45 ± 0.93 30.48 ± 0.23
(g/L)
C/N 10.23 ± 0.53 44.43 ± 0.92 80.44 ± 1.23 17.37 ± 0.45 24.75 ± 0.67 45.02 ± 0.66 14.34 ± 0.26 21.21 ± 0.43 30.43 ± 1.02
Total 9799.79 ± 80.34 22,36.01 ± 31.57 3731.50 ± 33.24 7494.43 ± 60.83 5833.70 ± 60.23 3857.76 ± 49.22 8011.47 ± 75.56 6081.93 ± 54.88 4581.04 ± 39.27
protein
concen-
tration
(mg/L)
Total 8666.32 ± 129.34 21,570.28 ± 306.44 6505.78 ± 89.35 9734.20 ± 150.73 13,221.36 ± 219.33 17,549.05 ± 283.76 7314.37 ± 113.55 7389.61 ± 102.34 7006.52 ± 104.31
starch
concen-
tration
(mg/L)
Total lipid 2835.33 ± 30.43 0.00 17,113.10 ± 291.34 1470.62 ± 20.34 1288.61 ± 18.20 703.05 ± 7.46 6249.22 ± 149.39 9688.46 ± 172.34 13,912.52 ± 281.44
concen-
tration
(mg/L)
Page 3 of 15
Ma et al. Biotechnol Biofuels (2017) 10:137
were as follows: total solids (TS) 54.90  ±  0.72  g/L, vol-
atile solids (VS)-to-TS ratio (VS/TS) 85.72  ±  1.23%,
protein 653.20  ±  10.01  mg/g TS, carbohydrate
208.41 ± 80.21 mg/g TS, and VFAs 3.45 ± 0.23 g/L. The
cultivated seeding sludge was washed three times with
deionized water before use in the fermentation reactors.
Experimental set‑up
Prior to fermentation, WAS was pretreated by heat and
alkali to accelerate the hydrolysis of organic matter. WAS
was heated at 105 °C for 3 h, and pH was adjusted to 12
with the addition of 5  M NaOH. A pH of 10 was main-
tained during fermentation by daily addition of 2 M HCl
or 2  M NaOH to inhibit the activity of methanogenic
bacteria. To ensure that enough acidogenic microor-
ganisms were in the fermentation reactor, an amount of
seeding sludge (~15% of VS) was added to control the
food–microorganism ratio (F/M) to about 5.67 before the
tests started. Nine 500-mL serum bottles were placed in
an air-bath shaker (35 ± 1 °C, 120 rpm) for anaerobic fer-
mentation. According to the substrate contents in each
bottle, they were labeled as OS (only pretreated sludge
as substrate), OP (only potato peel waste as substrate),
OF (only food waste as substrate), SP1 (VS ratio of pre-
treated sludge and potato peel waste = 3:1), SP2 (VS ratio
of pretreated sludge and potato peel waste = 1:1), SP3 (VS
ratio of pretreated sludge and potato peel waste  =  1:3),
SF1 (VS ratio of pretreated sludge and food waste = 3:1),
SF2 (VS ratio of pretreated sludge and food waste = 1:1),
SF3 (VS ratio of pretreated sludge and food waste = 1:3).
The total VS concentration of organic substrates before
fermentation was set to about 30  g/L and the fermenta-
tion was conducted in batch operation for 12  days. The
experimental set-up, as well as the initial organic matter
concentrations, are shown in Table  2. Before acidogenic
fermentation, 400 mL of substrate and seeding sludge was
added into each reactor and 50  mM 2-bromoethanesul-
fonic acid sodium salt was added to inhibit methanogens.
Each reactor was sealed with a rubber stopper and nitro-
gen was purged for 15 min to displace oxygen in the head-
space. The nitrogen purge was conducted at the beginning
of each fermentation and during each sample collection.
Analytical methods
All samples collected during fermentation were 10.0 mL
in volume, and were analyzed immediately after collec-
tion. Determination of TS, VS, soluble chemical oxygen
demand (SCOD), total COD (TCOD), and total nitrogen
(TN) were conducted according to standard methods
[18]. Total lipid and total starch were analyzed according
to Chinese standard methods [19]. Protein concentra-
tions were measured using the Lowry-Folin method [20].
The C/N ratio was calculated through the ratio of total
Page 4 of 15
organic carbon (TOC) to TN. TOC was detected with a
TOC analyzer (LiquiTOC, Elementar Analysensysteme,
Langenselbold, Germany). To measure soluble COD,
samples were first centrifuged at 10,200×g for 10  min,
and then filtered with 0.45-μm syringe filters.
VFAs concentrations in the filtrate samples were
detected by gas chromatography (GC-2010, Japan)
with flame ionization detection (FID). Separation was
achieved on a fused-silica capillary column (PEG-20M,
30 m × 0.32 mm × 0.5 mm, China). The initial column
temperature was 80 °C, and then it was heated to 210 °C
and held for 2 min. The running temperature was main-
tained at 80 °C during detection. The injection port and
detector temperatures were 250  °C. Before GC meas-
urement, 4-methyl-valeric acid (internal standard), 3  M
phosphoric acid (acidifier), and the filtrate sample were
mixed in a 1:1:1 ratio (v:v:v). This method was the same
as used in previous studies [21].
Calculations of the hydrolysis and acidification effi-
ciencies were performed according to the following
equations:
Hydrolysis efficiency (%)
(1)
= (SCODfinal − SCODinitial )/total COD
Acidification efficiency (%) = CODVFA /total COD,
(2)
where ­SCODfinal is the SCOD at the end of the fermen-
tation, mg/L; ­SCODinitial is the SCOD before fermenta-
tion, mg/L; and ­CODVFA is VFA concentration as COD
concentration, mg COD/L. The transformation of VFA to
COD was conducted as in previous studies [22].
DNA extraction
Samples of OS, OP, OF, SP3, and SF3 were preserved at the
end of fermentation for microbial analysis. The total DNA
of the five samples was extracted with a MoBio PowerSoil
DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA,
USA) according to the manufacturer’s instruction, and
as used in a previous study [23]. The quantity and qual-
ity of the extracted DNA were checked with a Nanodrop
2000 spectrophotometer (Thermo Scientific, Schaum-
burg, IL, USA). The two primers used for sequencing
on the Illumina Miseq sequencing platform were 515F
(5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGT-
CAATTCMTTTRAGTTT-3′). The raw sequence data
were deposited in the NCBI Sequence Read Archive (SRA)
database, and the accession number is SRP091729.
Canonical correspondence analysis
The relationship between bacteria at genus level and
environmental parameters was assessed with canoni-
cal correspondence analysis (CCA) using CANOCO 4.5
Ma et al. Biotechnol Biofuels (2017) 10:137
software. CCA is an effective method to combine envi-
ronmental factors to assess how the environment affects
research objects (such as bacteria). The arrows in CCA
represent environmental factors, and the four quadrants
indicate the positive and negative correlation between
the environmental factors and the two axes. The length
of the arrow represents the connection of environmental
factors and the distribution of the samples. For example,
longer arrow length indicates a greater influence of envi-
ronmental factors on the bacteria.
Results and discussion
VFAs production and composition during fermentation
Ethanol and four VFAs (acetate, propionate, butyrate,
and valerate) were observed in all fermentation groups
Fig. 1  The daily changes of ethanol and four single VFA concentrations during co-fermentation of WAS and potato peel waste (a), and WAS and
food waste (b)
Page 5 of 15
(Fig. 1a). In all cases, acetate was produced in large quan-
tities, making up 41.3–47.7% of VFAs content. When
fermented with only potato peel waste (OP), the acetate
concentration was 1.93  g/L. With an increased potato
peel waste addition ratio, the concentration of propi-
onate decreased dramatically from 1.11 to 0.32  g/L and
valerate decreased from 1.52 to 0.29 g/L. In contrast, the
concentration of ethanol increased from 0.33 to 1.08 g/L
and butyrate increased from 0.40 to 1.07 g/L. As a result,
the final percentages of acetate, ethanol, butyrate, propi-
onate, and valerate were 46.6, 6.7, 10.9, 16.9, and 18.9%,
respectively, in the OS group, and 41.3, 22.6, 21.3, 7.8,
7.0%, respectively, in the OP group.
As shown in Table  1, protein, starch, and lipid were
the main organic compounds in WAS, potato peel waste
Ma et al. Biotechnol Biofuels (2017) 10:137
and food waste, respectively. The addition of potato
peel waste resulted in increased starch concentration,
decreased protein concentration, and enhanced the
generation of ethanol and butyrate, while reducing the
accumulation of propionate and valerate. Some previ-
ous studies reported that when straw [24] or pretreated
bagasse [25] was used as co-fermentation substrate, the
variation of propionate and butyrate showed a similar
trend to that observed in this study. It is likely that the
polysaccharides were eventually biodegraded to glucose,
similar to the starch substrates used in the present study.
It has been verified that glucose fermentation above pH
6.0 produces butyrate and acetate as the main VFA prod-
ucts, while the dominant product is usually ethanol for
fermentation under more acidic conditions [26]. This
explanation is consistent with the increased accumula-
tion of butyrate and ethanol observed in this study.
As shown in Fig.  1b, when sludge was co-fermented
with food waste, acetate was also the dominant VFA
produced, with VFAs content ranging between 48.0 and
57.6%, although its concentration decreased significantly
to 2.68  g/L in the OF group. As the food waste addi-
tion ratio increased, valerate and propionate contents
gradually decreased from 1.10 to 0.41 and from 0.86 to
0.49  g/L, respectively. However, ethanol concentration
increased from 0.26 to 0.74  g/L and butyrate was rela-
tively stable (0.45–0.51 g/L).
Table  2 shows that the lipid concentration increased
from 2835.33  ±  30.43  mg/L in the OS group to
17113.10 ± 291.34 mg/L in the OF group as the ratio of
food waste increased, while the valerate and propion-
ate concentrations decreased and ethanol increased.
Lipids are mainly biodegraded to fatty acids and glycerol,
and glycerol further promotes the generation of ­H2 and
ethanol [27]. This might be a reason for the enhanced
ethanol generation in the present study. Decreased con-
centrations of valerate and propionate were observed
when potato peel waste and food waste were added,
and these changes might be related to low protein con-
centration. Several reports have suggested that nitro-
gen-rich substrates enhance propionate and valerate
generation during fermentation under alkaline condi-
tions. For example, increased protein consumption
Table 3  Extent of hydrolysis, acidification, and VFAs yields at the end of fermentation
Parameters OS SP1 SP2
Hydrolysis 49.67 ± 1.67 45.44 ± 1.02 51.17 ± 1.62
efficiency (%)
Acidification 14.51 ± 0.53 17.75 ± 0.68 21.25 ± 0.43
efficiency (%)
VFAs yield 132.30 ± 5.43 151.60 ± 6.83 268.40 ± 10.45
(mg COD/g VS)
Page 6 of 15
would promote propionate generation, and propionate,
instead of acetate, was the main product and accounted
for 63.4% of total VFAs in the co-fermentation of sludge
and food waste [9].
As shown in Fig. 1a and b, acetate was always the major
VFA produced, regardless of the composition of the
organic matter in the substrate. We speculate that there
are two possible reasons for the dominant acetate accu-
mulation. First, the seeding sludge used in this study was
domesticated for a long time before the fermentation,
and this would have enriched the acidogenic microorgan-
isms in the seeding sludge [28]. Second, alkaline condi-
tions in the fermentation killed or inhibited most of the
regular fermentative microorganisms, while the acido-
genic bacteria, with most being Gram-positive species
and able to generate spores, survived under the harsh
alkaline environment [29].
Organic matter changes during fermentation
Table  3 shows the extent of hydrolysis and acidifica-
tion, as well as the VFAs yields at the end of each acido-
genic fermentation. With the increasing ratio of potato
peel waste or food waste, hydrolysis efficiency gradu-
ally increased. In the nine fermentation groups, the two
highest hydrolysis efficiencies were observed in the OP
and OF groups (69.49 ± 1.91 and 54.01 ± 1.34%, respec-
tively). Potato peel waste and food waste were more eas-
ily hydrolyzed than WAS, thus improving hydrolysis
efficiency.
For acidification efficiency and VFAs yields, the same
trends were observed in all fermentation groups. When
potato peel waste was used as substrate for co-fermenta-
tion, the highest acidification efficiency (26.92  ±  0.46%)
and VFAs yield (343.54  ±  14.63  mg COD/g VS) were
observed in the SP3 group. With the addition of food
waste, the SF3 group presented the highest acidi-
fication efficiency (23.00  ±  0.64%) and VFAs yield
(282.02 ± 6.35 mg COD/g VS). The results demonstrated
that co-fermentation with sludge and carbon-rich sub-
strates benefited VFAs generation.
Comparing with Table 4, the VFAs yields in this study
were similar to those of Jia et al. [10], Rughoonund et al.
[25], and Huang et  al. [11], which gave the respective
SP3 OP SF1 SF2 SF3 OF
56.39 ± 1.53 69.49 ± 1.91 50.35 ± 1.38 48.45 ± 1.82 53.13 ± 1.69 54.01 ± 1.34
26.92 ± 0.46 16.74 ± 0.28 18.23 ± 0.27 20.62 ± 035 23.00 ± 0.64 17.56 ± 0.43
343.54 ± 14.63 185.10 ± 7.49 139.67 ± 5.32 217.85 ± 8.47 282.02 ± 6.35 182.52 ± 5.24
Table 4  Comparison of substrates, products, and microbial communities between previous literature studies and this study
Authors Type of substrates Target Operation Fermentation Highest Major Relative studies (Y to yes, N to no)
product mode condition product yield microorganism
Substrates Microbial
composition and  community and 
microbial community metabolic pathway
Ma et al. Biotechnol Biofuels (2017) 10:137

Jia et al. [10] WAS, perennial VFAs Batch 35 ± 1 °C, without pH 368.71 ± 17.53 Clostridia, Spirochaetes, Y N
ryegrass control gCOD/kg TS and Bacteroidetes
Rughoonundun WAS, bagasse VFAs Batch 55 °C, pH 7.0 360 mg/g VS – N N
et al. [25]
Huang et al. [11] WAS, henna plant VFAs Batch 35 ± 1 °C, initial pH 7891 ± 411 mg – N N
biomass 8.0, without pH COD/L
control
Guo et al. [30] WAS, agricultural VFAs Semi-continuous pH 10.0 ± 0.5 486.6 mgCOD/g VSS Proteobacteria, Firmicutes, Y N
residues and Actinobacteria
Huang et al. [31] WAS, bio-surfactants VFAs Batch pH 9.0, 10.0, and 11.0 425.2 mg COD/g VSS Firmicutes, Proteobacteria, Y N
Bacteroidetes, Actino-
bacteria, and Chloroflexi
Maspolim et al. [32] WAS VFAs Semi-continuous 35 °C, pH 4, 5, 6, 7, 8, 9, – Tissierella, Petrimonas, Y N
10, and 11 Proteiniphilum, Levilinea,
Proteiniborus, and
Sedimentibacter
Sivagurunathan Galactose H2 Batch and 35 ± 1 °C, pH over 5.5 1.05 mol H
­ 2/mol Clostridium sp. and Y Y
et al. [33] continuous galactose Sporolactobacillus sp.
Kumar et al. [34] Microalgae H2 Batch The seed inoculum 29.5  mL/g ­VSadded – N N
included BESA addi-
tion (1 g/L), pH 5.5
Ho et al. [35] Microalgal C. vulgaris Ethanol Batch 30 °C, pH 5.0-6.0 11.66 g/L Pure culture of Z. mobilis N N
FSP-E ATCC 29191
This study WAS, potato waste, VFAs Batch 35 ± 1 °C, pH 10.0 343.54 ± 14.63 mg Firmicutes, Chloroflexi, Y Y
food waste COD/g VS and Proteobacteria
– Not mentioned in literature
Page 7 of 15
Ma et al. Biotechnol Biofuels (2017) 10:137
figure of 368.71  ±  17.53  g COD/kg TS, 360  mg/g VS,
and 7891 ± 411 mg COD/L. However, the studies of Guo
et al. [30] and Huang et al. [31] gave higher VFA yields of
486.6  mg COD/g VS and 425.2  mg COD/g VS, respec-
tively, possibly a reflection of the different substrate
types. The VFAs yield in the OS group of the present
study was only 132.30 ± 5.43 mg COD/g VS, while previ-
ous studies using sludge-only fermentation showed VFAs
yields of 100–250  mg COD/g VS [36, 37]. The complex
composition of the sludge organics may be a reason for
the relative low VFAs generation. The low hydrolysis effi-
ciency was the limiting step of acidogenic fermentation,
especially in the sludge-only fermentation group (extent
of WAS hydrolysis  <50%). It is also noted that the fer-
mentation with the highest hydrolysis efficiency (OP) did
not show the highest VFAs production, indicating some
inconsistency between hydrolysis efficiency and acidifica-
tion efficiency.
The effects of potato peel waste or food waste on the
consumption of different organic matters at the end of
fermentation are shown in Fig.  2. When the addition
ratio of potato peel waste or food waste was below 50%,
protein, starch, and lipid consumption efficiencies were
higher than the WAS and carbon-rich substrate fermen-
tations (OS, OP, and OF). For example, the protein con-
sumption of SP1, SP2, SF1, and SF2 groups were 48.02,
50.16, 42.92, and 45.50%, respectively, which were higher
than the protein consumption in the WAS and carbon-
rich substrate fermentations (40.10% for OS, 17.28% for
OP, 19.75% for OF). It is likely that the appropriate addi-
tion ratio of carbon-rich substrates could be used to
enhance the consumption of organic matter. In addition,
a balance of nutrients in fermentation substrates could
Fig. 2  Consumption of different organic materials in the nine fer-
mentation groups
Page 8 of 15
be used to promote microbial growth and enhance the
activity of relevant enzymes. Feng et  al. [38] found that
addition of carbohydrate substrate promoted the con-
sumption of protein in sludge and promoted VFAs gen-
eration because of the activation of enzymes involved in
protein hydrolysis.
When the addition of potato peel waste or food waste
exceeded 50%, protein consumption decreased dramati-
cally (from 50.16 ± 0.88% in SP2 to 17.28 ± 0.09% in OP
and from 45.50 ± 0.45% in SF2 to 19.75 ± 0.10% in OF).
However, starch and lipid consumption were enhanced
significantly when the addition of potato peel waste or
food waste was increased to 75%. This demonstrated that
the consumption of carbon-rich substrate predominated
over protein consumption in a carbon-rich environment.
Moreover, starch and lipid consumption in potato-only
and food waste fermentation (OP and OF groups) were
less than in SP3 and SF3 groups. Evidently, unbalanced
nutrient supply was not beneficial for organic matter
consumption.
Relationship between substrate consumption and VFA
generation
The relationships between the generation of four dif-
ferent VFAs and the consumption of protein, lipids,
and starch at the end of each fermentation were further
analyzed. Figure  3a, d, and g shows that the accumula-
tions of propionate, butyrate, and valerate demonstrated
quadratic relations with lipid consumption, with the
R2 values of 0.63, 0.62, and 0.69, respectively. The gen-
eration of propionate and valerate was enhanced but
butyrate production was decreased when more lipids
were consumed. However, the trends in VFAs genera-
tion reversed when the lipid concentration exceeded
4000  mg/L. These results indicate that moderate lipid
consumption promoted the growth of acidogenic micro-
organisms, while excess lipid consumption inhibited it.
This caused more propionate and valerate accumula-
tion at a lower lipid consumption rate and less propi-
onate and valerate accumulation with increased lipid
consumption [39, 40]. Figure  3b, e, and h shows that
the generation of propionate and valerate gradually
decreased with starch consumption, but butyrate con-
centration proportionally increased. In relation to pro-
tein consumption, propionate and valerate accumulated
gradually (Fig.  3c, i) with higher protein consumption,
while butyrate concentration declined gradually at low
protein consumption, but then increased greatly (Fig. 3f )
at higher protein consumption.
According to the above results, starch was mainly
responsible for butyrate formation, while lipid and protein
consumption favored the synthesis of valerate and propion-
ate. However, different kinds of substrates can be converted
Ma et al. Biotechnol Biofuels (2017) 10:137 Page 9 of 15

Fig. 3  Plots and mathematic relationships between VFAs accumulation and consumption of lipids, starch, and protein during fermentation. a–c
Propionate accumulation; d–f butyrate accumulation; g–i valerate accumulation

to acetate by acidogenic microbes through different meta- Diversity of bacterial community

bolic pathways. Therefore, acetate accumulation became
dominant regardless of the composition of the substrates
in acidogenic fermentation under alkaline conditions. The
results in Additional file 1 further implied that pure protein
substrate (BSA group) gave a VFA distribution similarity
to that of the WAS fermentation group, while the glucose
(typical carbohydrate) substrate in acidogenic fermentation
gave a VFA distribution similarity to that of the potato-only
fermentation group. This demonstrated that fermentation
with glucose mainly produced acetate and butyrate while
fermentation with bovine serum albumin (BSA) preferen-
tially improved valerate and propionate production, apart
from the acetate accumulation.
As a measure of bacterial diversity, the numbers of shared
operational taxonomic units (OTUs) among OS, SP3,
OP, SF3, and OF fermentation groups were calculated by
Mothur’s Venn diagram analysis (Fig.  4a). Because most
bacteria could not survive in an alkaline fermentation
environment, a low OTU number of 733 was observed in
this study [32]. The total OTUs were 659, 464, 354, 130,
and 77 in OS, SP3, OP, OF, and SF3 samples, respectively.
Only 20 OTUs (2.7%) were shared by the five samples,
indicating that there was an obvious bacterial variety
over the five groups. OS, SP3, and OP groups shared 28
OTUs (4.2% of OS, 6.0% of SP3, and 36.4% of OP); OS,
SF3, and OF shared 34 OTUs (5.2% of OS, 9.6% of SF3,
Ma et al. Biotechnol Biofuels (2017) 10:137
Fig. 4  OTUs and the bacteria phylum distribution at the end of
fermentation. a Venn diagram analysis of the OS, OP, OF, SP3, and SF3
experiment groups; b PCA of the OS, OP, OF, SP3, and SF3 experiment
groups
and 26.2% of OF). The number of OTUs in the OS group
was the highest of the five fermentation groups, indicat-
ing that the OS sample had the most abundant microbial
populations. The Shannon–Weaver index of the OS sam-
ple (4.09) was also the highest among all the groups (2.25
for SP3, 1.90 for SF3, 1.98 for OP, and 2.40 for OF). This
was because of the relative abundance of protein in the
OS sample when compared with SP3, OP, SF3, and OF,
which would lead to the survival of more protein-con-
suming bacteria.
Page 10 of 15
Principal component analysis (PCA) of classified OTUs
revealed that the microbial communities in SP3, OP, SF3,
and OF fermentation groups were significantly shifted
from that in the OS group (Fig.  4b). Relatively similar
communities occurred in OP and SP3, and in OF and
SF3, but not among the four tests. This finding was fur-
ther supported by the results of taxonomic analysis. The
different classification groups in the five groups clearly
revealed the significant impact of organic matter compo-
sition on microbial similarity during fermentation.
The distribution of the bacterial community further
explained the differences among OS, OF, OP, SF3, and
SP3 groups in detail (Fig.  5a). Five groups showed rela-
tively low diversities with a total of 7 identified phyla
observed. The phyla Firmicutes, Chloroflexi, and Pro-
teobacteria, which were recognized as common fermen-
tative phyla [41], were dominant in all five communities
with a combined contents of 84.0, 98.9, 99.7, 98.1, and
97.5% in OS, OF, OP, SF3, and SP3, respectively. How-
ever, the distribution of the three phyla in the five groups
was obviously different. Firmicutes showed the highest
relative abundance in the five groups. From Table 4, Fir-
micutes, Chloroflexi, and Proteobacteria could always be
enriched under alkaline conditions, and these bacteria
mainly participated in VFAs generation.
At the class level, it was observed that Clostridia
(28.2%), Bacilli (11.7%), Gammaproteobacteria (10.0%),
Synergistia (7.9%), and Anaerolineae (7.8%) were the five
main classes in the OS group, while Bacilli and Clostridia
were the two major classes in the SP3, OP, SF3, and OF
groups. The abundance of the Bacilli class increased dra-
matically when potato peel waste was added in the sludge
(72.2% in SP3 and 64.1% in OP), while the Clostridia class
rose significantly with the addition of food waste (56.2%
in SF3 and 53.6% in OF). Some studies have reported that
bacteria from Clostridia and Bacilli were able to produce
acetate; however, Clostridia bacteria can also produce
butyrate [42], and Bacilli bacteria can produce lactic acid
[43]. The distribution of special bacterial class in different
fermentation groups clearly proved that the composition
of organic matter could significantly influence the bacte-
rial community structure, and selectively enrich specific
acidogenic bacteria during anaerobic co-fermentation.
To further explore the microbial diversity in the five
groups, the results of hierarchical clustering analysis at
the genera level are shown in Fig. 5b (genera with relative
abundance >1% in each sludge sample are listed in Addi-
tional file 2). Relatively similar communities occurred in
groups SP3 and SF3, and in OP and OF. Nevertheless, the
bacterial community in the OS group was different from
the other four groups. This further proves that the com-
position of organic matter influences and shifts the bac-
terial community structure at genus level.
Ma et al. Biotechnol Biofuels (2017) 10:137 Page 11 of 15

Fig. 5  Taxonomic classification of sequences. a Bacterial communities of the OS, OP, OF, SP3, and SF3 experiment groups at class level and phylum
level. b Hierarchical clustering analysis at genus level of bacterial communities of the OS, OP, OF, SP3, and SF3 fermentation groups

Relationship between substrate composition and bacteria
community
Previous studies have explored the relationship between
microbial activity and different types of substrates in
co-fermentation (Table  4). For example, Guo et  al. [30]
found that bacteria like Proteobacteria and Firmicutes
were enriched under alkaline conditions with added agri-
cultural residue. Moreover, use of perennial ryegrass or
fermentation with only WAS gave different microbial
communities [10]. Several researchers have also stud-
ied different microbial communities and their meta-
bolic pathways based on a single type of substrate. For
example, Sivagurunathan et  al. [33] reported that two
Clostridium strains showed different metabolic pathways
in different operation modes with galactose as substrate.
However, little attention was paid to the relationship
between the organic matter composition and the micro-
bial mechanism, or details such as the microbial commu-
nity and metabolic pathway.
To explore how the organic matter composition in
solid wastes influenced the structure of the bacterial
community in detail, the relationship between organic
matter composition and typical genera was explained
by CCA (Fig.  6). The typical genera of Anaerobacillus,
Clostridium, Amphibacillus, and so on, were all located
close to the starch, indicating that these genera could be
enriched by the feedstock with high starch content (SP3
and OP). With increased lipid content in the feedstock,
the dominant genera like Hafnia, Brochothrix, and Leu-
conostoc were enriched in SF3 and OF samples. In the
OS sample, the dominant genera like Hyphomicrobium,
Brassicibacter, Gracilibacter, Ornatilinea, and SRB2 were
Ma et al. Biotechnol Biofuels (2017) 10:137
Fig. 6  CCA analysis of the microbial communities and the organic
composition between the OS, OP, OF, SP3, and SF3 fermentation
groups
more likely to be enriched, indicating that these genera
probably had a close relationship with protein degrada-
tion. These results indicated that the structure of the
bacterial community was significantly influenced by the
organic components in the substrates and thereby gener-
ated particular products. For example, the Clostridiales,
which was dominant in the starch-enriched substrate,
contributed significantly to butyrate production [44],
while Anoxybacillus, a kind of anaerobic bacteria that
only consumes carbohydrate, produced formate, lactate,
acetate, and ethanol in low concentrations [45]. Ornati-
linea, which was abundant in the OS fermentation group,
was able to consume a variety of protein substrates to
produce valerate as the main product [46]. The intersec-
tion angle between starch and canonical axis 1 (Fig.  6)
was smaller than the factors of lipids and protein, sug-
gesting that starch was more important than other fac-
tors in determining the typical genera in the five groups.
The close relationship between protein and the bacterial
community mainly explained the enrichment of some
specific genera in the OS group.
To further investigate how the different substrates
determined the VFAs distribution, the VFAs synthe-
sis pathway was analyzed in detail (Fig. 7). Lipid, starch,
and protein are transformed to pyruvate as the common
intermediate through a series of biochemical reactions,
and then transformed into downstream intermediates.
Therefore, pyruvate can be considered as the core and
starting point of the VFAs metabolic pathway network
Page 12 of 15
with different substrates. The metabolic pathways and the
theoretical chemical equations for propionate, butyrate,
and valerate are listed in Fig. 7.
Some propionate-producing bacteria, like Planctomyc-
etaceae and Brassicibacter, were enriched by consuming
a large amount of protein (Fig.  6). From Fig.  7a, propi-
onate was generated through two steps from pyruvate,
and vitamin B12 was a co-factor to carboxyl transferase
of methyl malonyl CoA, which was involved in the con-
version step of pyruvate to malonyl CoA. Vitamin B12
could not exist alone in strong acidic or alkaline condi-
tions unless it combined with protein [47], and the lack of
protein would limit the combination of vitamin B12, thus
inhibiting propionate generation. The presence of vita-
min B12 might benefit propionate accumulation.
Valerate production was related to the conversion of
glutamate, which was generated from protein (Fig.  7b).
Bacteria like Bacillus reduced glutamate to proline [48]
and then further reduced it to 5-aminovalerate. Finally,
5-aminovalerate was fermented by bacteria like Brassici-
bacter via 5-hydoxyvaleryl-CoA and 2-pentenoyl-CoA
to valerate. Ammonia, acetate, and propionate were also
produced simultaneously during fermentation. The path-
way in Fig. 7b also demonstrates that the consumption of
protein could promote the generation of acetate, propi-
onate, and valerate.
The increase in butyrate that was observed with the
addition of starch is possibly related to the type of
butyrate-producing bacteria and the acetate concentra-
tion (Fig. 7c). It was reported that most butyrate-produc-
ing bacteria preferred glucose as their substrates [49], and
the enzymes related to butyrate production were mainly
transferases of acetyl-coenzyme and butyrate kinase.
Over 50% of butyrate-producing bacteria, like Clostrid-
ium sensu stricto and Brochothrix, have both enzymes,
which means that they could produce butyrate through
the transformation of intracellular acetate [50]. However,
some butyrate-producing bacteria could only generate
butyrate by using extracellular acetate because of the
absence of butyrate kinase. In this study, the SP3 and OP
groups had abundant acetate and high concentrations of
glucose as extracellular substrates for butyrate-producing
bacteria, thus offering a good environment for butyrate
generation.
Bacteria related to acetate production were extensively
distributed in the fermentation. For example, bacteria like
Ornatilinea and Gracilibacter could produce acetate by
consuming the protein or carbohydrate substrates. More-
over, the process of acetate generation and conversion are
complex. Figure 7d shows that acetate was generated not
only by the conversion of acetyl-coenzyme A, but also
through ­H2 and ­CO2 transformation by homoacetogene-
sis and acetaldehyde transformation by Saccharomycetes
Ma et al. Biotechnol Biofuels (2017) 10:137 Page 13 of 15

Fig. 7  The synthetic pathways of propionate (a), valerate (b), butyrate (c), and acetate (d). Dotted arrows represent the transport pathway of sub-
strate from extracellular environment to intracellular cytoplasm; solid arrows represent the synthetic pathway of single VFA. Arrows in different colors
represent different synthetic pathways
Ma et al. Biotechnol Biofuels (2017) 10:137
in the VFA pathway [51]. The extensive distribution of
acetate-producing bacteria and their various metabolic
pathways made acetate the major VFA produced during
acidogenic fermentation.
Conclusion
Substrate composition significantly influenced the VFAs
production and distribution in acidogenic co-fermen-
tation, as observed by the addition of different types of
organic matter. In all cases, acetate was the dominant
product in acidogenic fermentation, regardless of the
substrate composition. The fermentation profiles and
metabolic pathway analysis revealed that the addition of
carbon-rich substrates significantly enhanced butyrate
and ethanol accumulation, while protein-rich substrates
substantially favored propionate and valerate generation.
The abundance of the Bacilli class increased dramati-
cally when potato peel waste was added to the sludge,
while the Clostridia class rose significantly with the addi-
tion of food waste. These trends indicate that the organic
matter composition significantly influenced the bacterial
community structure, and selectively enriched specific
acidogenic bacteria during anaerobic fermentation. The
novel findings in this study are very helpful for substrate
selection and parameter control for future selective VFAs
production by acidogenic co-fermentation from waste
activated sludge.
Additional files
Additional file 1. Single VFA accumulation at the end of alkaline fermen-
tation with glucose and bovine serum albumin (BSA).
Additional file 2. Content of bacteria at genus level in OS, SP3, SF3, OP,
and OF fermentation groups (data given as percentages).
Abbreviations
BSA: bovine serum albumin; CCA: the canonical correspondence analysis;
COD: chemical oxygen demand; C/N: carbon-to-nitrogen ration; FID: flame
ionization detector; GC: gas chromatograph; OF: the fermentation substrate
was only food waste; OP: the fermentation substrate was only potato peel
waste; OS: the fermentation substrate was only pretreated sludge; OTUs: oper-
ational taxonomic units; PCA: principal component analysis; SF1: the VS ratio
of pretreated sludge and food waste was 3:1; SF2: the VS ratio of pretreated
sludge and food waste was 1:1; SF3: the VS ratio of pretreated sludge and food
waste was 1:3; SP1: the VS ratio of pretreated sludge and potato peel waste
was 3:1; SP2: the VS ratio of pretreated sludge and potato peel waste was 1:1;
SP3: the VS ratio of pretreated sludge and potato peel waste was 1:3; TN: total
nitrogen; TS: total solid; VFAs: volatile fatty acids; VS: volatile solid; WAS: waste
activated sludge.
Authors’ contributions
HL designed the study, analyzed the results, and revised the manuscript. HM
performed the experiments and wrote the manuscript. LZ and MY took part
in the experimental work. BF helped analyze the data of bacteria community
and revise the manuscript. HL participated in most of data analysis. All authors
discussed the results and helped to draft the manuscript. All authors read and
approved the final manuscript.
Page 14 of 15
Author details
1
 School of Environmental and Civil Engineering, Jiangnan University,
Wuxi 214122, China. 2 Jiangsu Key Laboratory of Anaerobic Biotechnology,
Wuxi 214122, China.
Acknowledgements
We would like to acknowledge Dr. Walquiria Simpcicio for her contribution to
the revision processes of this manuscript. This research was financially sup-
ported by the National Natural Science Foundation of China (51678280), the
National Key Technology R&D Program of the Ministry of Science and Technol-
ogy (2014BAD24B03-02), the Fundamental Research Funds for the Central
Universities (JUSRP51633B), and the Natural Science Foundation of Jiangsu
Province of China (BK20141112).
Competing interests
The authors declare that they have no competing interests.
Availability of supporting data
The authors promise the availability of supporting data.
Consent for publication
The authors have consented for publication.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 19 March 2017 Accepted: 17 May 2017
References
1. Nishio N, Nakashimada Y. Recent development of anaerobic diges-
tion processes for energy recovery from wastes. J Biosci Bioeng.
2007;103:105–12.
2. Appels L, Baeyens J, Degrève J, Dewil R. Principles and potential of the
anaerobic digestion of waste-activated sludge. Prog Energy Combust Sci.
2008;34:755–81.
3. Wu H, Gao J, Yang D, Zhou Q, Liu W. Alkaline fermentation of primary
sludge for short-chain fatty acids accumulation and mechanism. Chem
Eng J. 2010;160:1–7.
4. Sheng GP, Yu HQ, Li XY. Extracellular polymeric substances (EPS) of micro-
bial aggregates in biological wastewater treatment systems: a review.
Biotechnol Adv. 2010;28:882–94.
5. Chen H, Wang D, Li X, Yang Q, Zeng G. Enhancement of post-anoxic
denitrification for biological nutrient removal: effect of different carbon
sources. Environ Sci Pollut Res. 2015;22:5887–94.
6. Andersen SJ, Candry P, Basadre T, Khor WC, Roume H, Hernandez-Sana-
bria E, et al. Electrolytic extraction drives volatile fatty acid chain elonga-
tion through lactic acid and replaces chemical pH control in thin stillage
fermentation. Biotechnol Biofuels. 2015;8:1–14.
7. Agler MT, Wrenn BA, Zinder SH, Angenent LT. Waste to bioproduct con-
version with undefined mixed cultures: the carboxylate platform. Trends
Biotechnol. 2011;29:70–8.
8. Liu H, Xiao H, Yin B, Zu Y, Liu H, Fu B, et al. Enhanced volatile fatty acid pro-
duction by a modified biological pretreatment in anaerobic fermentation
of waste activated sludge. Chem Eng J. 2016;284:194–201.
9. Chen Y, Luo J, Yan Y, Feng L. Enhanced production of short-chain fatty
acid by co-fermentation of waste activated sludge and kitchen waste
under alkaline conditions and its application to microbial fuel cells. Appl
Energy. 2013;102:1197–204.
10. Jia S, Dai X, Zhang D, Dai L, Wang R, Zhao J. Improved bioproduction of
short-chain fatty acids from waste activated sludge by perennial ryegrass
addition. Water Res. 2013;47:4576–84.
11. Huang J, Zhou R, Chen J, Han W, Chen Y, Wen Y, et al. Volatile fatty acids
produced by co-fermentation of waste activated sludge and henna plant
biomass. Bioresour Technol. 2016;211:80–6.
Ma et al. Biotechnol Biofuels (2017) 10:137
12. Liu X, Li R, Ji M, Han L. Hydrogen and methane production by co-
digestion of waste activated sludge and food waste in the two-stage
fermentation process: substrate conversion and energy yield. Bioresour
Technol. 2013;146:317–23.
13. Zhang J, Lv C, Tong J, Liu J, Liu J, Yu D, Wang Y, et al. Optimization and
microbial community analysis of anaerobic co-digestion of food waste
and sewage sludge based on microwave pretreatment. Bioresour Tech-
nol. 2016;200:253–61.
14. Yang M, Kuittinen S, Zhang J, Vepsäläinen J, Keinanen M, Pappinen A.
Co-fermentation of hemicellulose and starch from barley straw and grain
for efficient pentoses utilization in acetone–butanol–ethanol production.
Bioresour Technol. 2015;179:128–35.
15. Silvestre G, Fernandez B. A. Bonmati. Addition of crude glycerine as
strategy to balance the C/N ratio on sewage sludge thermophilic and
mesophilic anaerobic co-digestion. Bioresour Technol. 2015;193:377–85.
16. Zhang W, Werner JJ, Matthew AT, Angenent LT. Substrates type drives
variation in reactor microbiomes of anaerobic digesters. Bioresour Tech-
nol. 2014;151:397–401.
17. Wang J, Liu H, Fu B, Xu K, Chen J. Trophic link between syntrophic aceto-
gens and homoacetogens during the anaerobic acidogenic fermentation
of sewage sludge. Biochem Eng J. 2013;70:1–8.
18. APHA. Standard methods for the examination of water and wastewater.
Washington DC: American Public Health Association Inc.; 1998.
19. SEPA. Water and wastewater monitoring methods. 4th ed. Beijing: Chi-
nese Environmental Science Publishing House; 2002.
20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with
the folin phenol reagent. J Biol Chem. 1951;193:265–75.
21. Ma H, Chen X, Liu H, Liu H, Fu B. Improved volatile fatty acids anaerobic
production from waste activated sludge by pH regulation: alkaline or
neutral pH? Waste Manag. 2016;48:397–403.
22. Liu X, Liu H, Chen Y, Du G, Chen J. Effects of organic matters and initial
carbon-nitrogen-ratio on the bioconversion of volatile fatty acids from
sewage sludge. J Chem Technol Biotechnol. 2008;83:1049–55.
23. Xu S, Fu B, Zhang L, He L. Bioconversion of ­H2/CO2 by acetogen enriched
cultures for acetate and ethanol production: the impact of pH. World J
Microbiol Biotechnol. 2015;31:941–50.
24. Zhou A, Guo Z, Yang C, Kong F, Liu W, Wang A. Volatile fatty acids produc-
tivity by anaerobic co-digesting waste activated sludge and corn straw:
effect of feedstock proportion. J Biotechnol. 2013;168:234–9.
25. Rughoonundun H, Mohee R, Holtzapple MT. Influence of carbon-to-
nitrogen ratio on the mixed-acid fermentation of wastewater sludge and
pretreated bagasse. Bioresour Technol. 2012;112:91–7.
26. Matsushika A, Sawayama S. Characterization of a recombinant flocculent
Saccharomyces cerevisiae strain that co-ferments glucose and xylose:
influence of pH and acetic acid on ethanol production. Appl Biochem
Biotechnol. 2012;168:2094–104.
27. Clomburg JM, Gonzalez R. Anaerobic fermentation of glycerol: a platform
for renewable fuels and chemicals. Trends Biotechnol. 2013;31:20–8.
28. Rosa PRF, Santos SC, Sakamoto IK, Varesche MBA, Silva EL. The effects of
seed sludge and hydraulic retention time on the production of hydrogen
from a cassava processing wastewater and glucose mixture in an anaero-
bic fluidized bed reactor. Int J Hydrog Energy. 2014;39:13118–27.
29. Yuan Y, Liu Y, Li B, Wang B, Wang S, Peng Y. Short-chain fatty acids produc-
tion and microbial community in sludge alkaline fermentation: long-term
effect of temperature. Bioresour Technol. 2016;211:685–90.
30. Guo Z, Zhou A, Yang C, Liang B, Sangeetha T, He Z, et al. Enhanced short
chain fatty acids production from waste activated sludge conditioning
with typical agricultural residues: carbon source composition regulates
community functions. Biotechnol Biofuels. 2015;8:192–206.
31. Huang X, Mu T, Shen C, Lu L, Liu J. Effects of bio-surfactants combined
with alkaline conditions on volatile fatty acid production and microbial
community in the anaerobic fermentation of waste activated sludge. Int
Biodeterior Biodegrad. 2016;114:24–30.
32. Maspolim Y, Zhou Y, Guo C, Xiao K, Ng WJ. The effect of pH on solubiliza-
tion of organic matter and microbial community structures in sludge
fermentation. Bioresour Technol. 2015;190:289–98.
Page 15 of 15
33. Sivagurunathan P, Kumar G, Park JH, Park JH, Park HD, Yoon JJ, Kim SH.
Feasibility of enriched mixed cultures obtained by repeated batch
transfer in continuous hydrogen fermentation. Int J Hydrog Energy.
2016;41:4393–403.
34. Kumar G, Zhen G, Sivagurunathan P, Bakonyi P, Nemestóthy N, Bélafi-Bakó
K, Kobayashi T, et al. Biogenic ­H2 production from mixed microalgae bio-
mass: impact of pH control and methanogenic inhibitor (BESA) addition.
Biofuel Res J. 2016;11:470–4.
35. Ho SH, Huang S, Chen C, Hasunuma T, Kondo A, Chang JS. Bioethanol
production using carbohydrate-rich microalgae biomass as feedstock.
Bioresour Technol. 2013;135:191–8.
36. Luo J, Feng L, Chen Y, Sun H, Shen Q, Li X, Chen H. Alkyl polyglucose
enhancing propionic acid enriched short-chain fatty acids production
during anaerobic treatment of waste activated sludge and mechanisms.
Water Res. 2015;73:332–41.
37. Kayhanian M, Tchobanoglous G. Computation of C/N ratios for various
organic fractions. BioCycle. 1992;33:58–60.
38. Feng L, Chen Y, Zheng X. Enhancement of waste activated sludge protein
conversion and volatile fatty acids accumulation during waste activated
sludge anaerobic fermentation by carbohydrate substrate addition: the
effect of pH. Environ Sci Technol. 2009;43:4373–80.
39. Phan S, Salentinig S, Gilbert E, Darwish TA, Hawley A, Nixon-Luke R,
Bryant G, Boyd BJ. Disposition and crystallization of saturated fatty acid
in mixed micelles of relevance to lipid digestion. J Colloid Interface Sci.
2015;449:160–6.
40. Yang J, Xiong YL. Inhibition of lipid oxidation in oil-in-water emul-
sions by interface-adsorbed myofibrillar protein. J Agric Food Chem.
2015;63:8896–904.
41. Regueiro L, Veiga P, Lema JM, Carballa M. Influence of transitional states
on the microbial ecology of anaerobic digesters treating solid wastes.
Appl Microbiol Biotechnol. 2014;98:2015–27.
42. Cho C, Jang YS, Moon HG, Lee J, Lee SY. Metabolic engineering of
clostridia for the production of chemicals. Biofuels Bioprod Biorefin.
2015;9:211–25.
43. Daffé M. The cell envelope of tubercle bacilli. Tuberculosis. 2015;95:155–8.
44. Levine UY, Looft T, Allen HK, Stanton TB. Butyrate-producing bacteria,
including mucin degraders, from the swine intestinal tract. Appl Environ
Microbiol. 2013;79:3879–81.
45. Goh KM, Kahar UM, Chai YY, Chong CS, Chai KP, Ranjani V, Illias RM, Chan
KG. Recent discoveries and applications of Anoxybacillus. Appl Microbiol
Biotechnol. 2013;97:1475–88.
46. Podosokorskaya OA, Bonch-Osmolovskaya EA, Novikov AA, Kolganova
TV, Kublanov IV. Ornatilinea apprima gen. nov., sp. nov., a novel cel-
lulolytic representative of class Anaerolineae. Int J Syst Evol Microbiol.
2012;63:86–92.
47. Savvi S, Warner DF, Kana BD, McKinney JD, Mizrahi V, Dawes SS. Functional
characterization of a Vitamin B12-dependent methylmalonyl pathway
in Mycobacterium tuberculosis: implications for propionate metabolism
during growth on fatty acids. J Bacteriol. 2008;190:3886–95.
48. Buckel W. Unusual enzymes involved in five pathways of glutamate
fermentation. Appl Microbiol Biotechnol. 2001;57:263–73.
49. Wiese BI, Górka P, Mutsvangwa T, Okine E, Penner GB. Short communica-
tion: interrelationship between butyrate and glucose supply on butyrate
and glucose oxidation by ruminal epithelial preparations. J Dairy Sci.
2013;96:5914–8.
50. Jang YS, Im JA, Choi SY, Lee JI, Lee SY. Metabolic engineering of Clostrid-
ium acetobutylicum for butyric acid production with high butyric acid
selectivity. Metab Eng. 2014;23:165–74.
51. Patil SA, Arends JBA, Vanwonterghem I, Meerbergen J, Guo K, Tyson GW,
Rabaey K. Selective enrichment establishes a stable performing commu-
nity for microbial electrosynthesis of acetate from ­CO2. Env Sci Technol.
2015;49:8833–43.